Project Summary An invaluable opportunity has emerged in advanced optical microscopy for obtaining accurate and precise measurements of distance, occupancy and dynamics in biologically important macromolecules. Frster resonance energy transfer (FRET) has been available for some time to measure distances in macromolecular assemblies that are labeled with fluorescent donor-acceptor probe pairs separated by ~2 ? 8 nm. However, artifacts and uncertainties arise in classical ensemble (cuvette) FRET experiments due to non-stoichiometric labeling, contaminants, anomalous photo-physical behavior, unknown rotational mobilities, and averaging over static and dynamic inhomogeneities. Moreover, inhomogeneities may represent normal functional variations or sample degradation. These problems are overcome in the requested instrument by simultaneously recording spectral properties of the probes as individual macromolecules diffuse through a microscopic volume. A single molecule multi-parameter fluorescence detection (smMFD) system with confocal detection (a) dramatically reduces quantitative uncertainties, (b) greatly increases accuracy, (c) is much faster, (d) greatly simplifies sample quantity and preparation requirements, and (e) ensures identical sample conditions because all measurements are made over a short time interval on the same sample. Sample immobilization is not required, which both simplifies sample preparation and eliminates artifacts. Two correlated detectors are required for the donor and two for the acceptor to eliminate detector dead time and after-pulsing. When a polarizing beam splitter projects the emission onto each pair of detectors, rotational anisotropy is also obtained in the same experiment. Therefore, each detection event informs an 8-fold parameter space consisting of anisotropy, lifetime, intensity (stoichiometry), detection time (related to diffusion coefficient), excitation spectrum, emission spectrum, quantum yield, and distance between the two fluorophores. An analysis of these signals enables the identification of individual species present in the population of molecules, and determination of the interconversion rates between these species. The multi-parameter fluorescence detection approach enables us to discriminate among heterogeneous species, correct for labeling stoichiometry, and quantify dynamic motions as the molecule explores its conformational space. Energy transfer efficiency is converted to the distance between the labeling sites by computational analysis of a dynamic structural model with tethered probes. The instrument requested in this proposal will make this powerful technology available to all interested investigators.